The overall aim of this application is to define parameters in trinucleotide repeats found in humans which confer instability and predisposition to disease. Mutant trinucleotide repeats have been implicated in seven human genetic disorders with neuromuscular effects to date. One of the hallmarks of these mutations is extreme mutability, i.e. the mutations confer hypermutability onto themselves. Mutations of the repeats are involve in altering the severity an/or onset of the disease. Therefore, the mechanisms involved in generating these novel mutations are quite relevant to the study of these disorders. This effort will test the hypothesis that instability in the CCGG repeat found at the FMR1 gene involved in fragile X syndrome is intrinsic to the repeat itself. The alternative hypothesis that other closely linked sequences are involved will also be tested. This will be accomplished through the following aims: 1) Identification of the molecular basis of meiotic instability using empirical studies of stable and unstable repeats found in human families. 2) Development of a model system capable of testing repeat elements for stability. 30 Study of similar repeats in the human and other species' genomes in order to compare and contrast these without the human FMRI repeat. 4) Study of the methylation of CGG repeats in and its role in stability of the repeat during early development. 5) Extension of these studies to other trinucleotide repeats, particularly the CAG repeat found to be unstable in several other human genetic disorders. 6) Provision of medically relevant samples to collaborators interested in the study of trinucleotide repeats using a variety of biochemical and enzymatic measures. Determination of the types of repeats (or other nearby sequences) involved in instability will provide for more accurate forecasting of the behavior of these elements and should allow for improved diagnostic testing. In addition, this information in combination with the studies of the other members of this program project will provide significant insight into the types of replication and/or repair errors underlying the behavior of these sequences. These data will provide new understanding of the basic enzymology and biochemistry underpinning DNA replication and repair in humans, with likely consequences for somatic mutation as well.